title: A multiplex qPCR gene dosage assay for rapid genotyping and large-scale population screening for deletional α-thalassemia.
abstract: The predominant determinants of α-thalassemia are deletions in the human α-globin gene cluster. A rapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human α-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six α-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-ΔΔCq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for α-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening.
authors.affiliation: Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.
authors.forename: Wanjun
authors.initials: W
authors.lastname: Zhou
authors.forename: Ge
authors.initials: G
authors.lastname: Wang
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Zhao
authors.forename: Fu
authors.initials: F
authors.lastname: Xiong
authors.forename: Shaoxiong
authors.initials: S
authors.lastname: Zhou
authors.forename: Jianming
authors.initials: J
authors.lastname: Peng
authors.forename: Youming
authors.initials: Y
authors.lastname: Cheng
authors.forename: Shun
authors.initials: S
authors.lastname: Xu
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Xu
mesh_terms: D018628:Gene Dosage; D023061:Gene Order; D005838:Genotype; D006801:Humans; D008403:Mass Screening; D058885:Multilocus Sequence Typing / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055542:alpha-Globins / Q000235:genetics; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D055542:alpha-Globins